Method for more efficient electroporation

ABSTRACT

This invention provides improved electroporation methods for transferring nucleic acids of interest into host cells, wherein the host cells are (1) suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative, (2) mixed with the nucleic acids of interest, and (3) electrically treated. Also, this invention provides for kits used in the method for transferring nucleic acids into host cells.

[0001] The invention relates to the field of electroporation andtransformation of host cells, particularly bacterial cells.

BACKGROUND OF THE INVENTION

[0002] Introducing nucleic acids into E. coli and other host organismsis central to many types of experiments and analyses. For example, whensearching for a gene of interest in a DNA library, the library must betransferred into a host organism. Since the DNA of many organisms isvery complex, the number of independent clones that are needed tocompletely represent the organism is large. In order to create a librarythat completely represents the organism, the efficiency at which the DNAcan be introduced into the host cell becomes limiting. By optimizingthis process, the ability to create and screen DNA libraries isfacilitated.

[0003] Similarly, many other experimental analyses are limited by theability to introduce DNA into a host organism. When cloning largesegments of DNA for whole genome analysis (i.e., using bacterialartificial chromosomes), when performing PCR cloning, or when carryingout random mutagenesis of a gene, followed by cloning all potentialaltered forms, success often depends on the size of the initialtransformation pool. Again, developing conditions that improve theprocess of introducing nucleic acids into a host organism increases thechance that the experiment will succeed.

[0004] There are several methods for introducing nucleic acids intovarious host cells, e.g., incubating the host cells with co-precipitatesof nucleic acids (Graham and van der Eb, Virology, 52: 456467 (1973)),directly injecting genes into the nucleus of the host cells (Diacumakos,Methods in Cell Biology, Vol. 7, eds. Prescott, D. M. (Academic Press)pp. 287-311 (1973), introducing nucleic acids via viral vectors (Hamerand Leder, Cell, 18: 1299-1302 (1979)), and using liposomes as a meansof gene transfer (Fraley et al., J. Biol. Chem., 255:10431-10435 (1980);Wong et al. Gene, 10: 87-94 (1980)). Electroporation has also been usedto transform host organisms, including E. coli. (Dower et al., NucleicAcids Research, 16: 6127-6145 (1988); Taketo, Biochimica et BiophysicaActa, 949: 318-324 (1988); Chassy and Flickinger, FEMS MicrobiologyLetters, 44: 173-177 (1987); and Harlander, Streptococcal Genetics, eds.Ferretti and Curtiss (American Society of Microbiology, Washington,D.C.) pp. 229-233 (1987)).

[0005] In general, electroporation involves the transfer of genes orgene fragments (nucleic acids) into a host cell by exposure of the cellto a high voltage electric impulse in the presence of the genes or genefragments (Andreason and Evans, Biotechniques, 6: 650-660 (1988)). Quiteoften, the genes and gene fragments are exogenous, i.e., heterologous tothe host organism. Also, frequently the cells have been stored prior toelectroporation. A typical method of storage is to freeze the cells. Thecells are frozen at a temperature that preserves viability. Afterthawing those cells, genes or gene fragments may be transferred byelectroporation into the cells, permanently or transiently forshort-term expression.

[0006] An example of a typical electroporation method is to growbacteria in enriched media (of any sort) and to concentrate the bacteriaby washing in a buffer that contains 10% glycerol (Dower et al., 1988,U.S. Pat. No. 5,186,800). As discussed in U.S. Pat. No. 5,186,800, whichis hereby incorporated by reference in its entirety, DNA is added to thecells and the cells are subjected to an electrical discharge, whichtemporarily disrupts the outer cell wall of the bacterial cells to allowDNA to enter the cells.

[0007] The electrical treatment to which the host cells are subjectedduring the process of electroporation is very harsh and typicallyresults in the death of >90% of the host cells. However, it is believedthat the majority of cells that survive electroporation take up thenucleic acids of interest. The efficiency with which nucleic acidtransfer occurs depends on a variety of factors, including the geneticbackground of the host cells. Routinely, an efficiency of 10⁹-1×10¹⁰transformants per μg of input DNA (plasmid pUC18) may be achieved. UsingRecA-cells, typically 5.0-7.0×10⁹ cells are transformed per μg of inputDNA. When the host cells are E. coli, 10% or less of the treatedbacteria survive. However, the percentage is significantly lower forcertain strains of E. coli that are inefficient atelectrotransformation.

[0008] In developing and refining electroporation methodology,researchers have identified factors that impact the efficiency of thetransfer. These factors include, e.g., the electrical field strength,the pulse decay time, the pulse shape, the temperature in which theelectroporation is conducted, the type of cell, the type of suspensionbuffer, and the concentration and size of the nucleic acid to betransferred (Andreason and Evans, Analytical Biochemistry, 180: 269-275(1988); Sambrook, et al., Molecular Cloning: a Laboratory Manual, 2ndEdition, eds. Sambrook, et al. (Cold Spring Harbor Laboratory Press) pp.1.75 and 16.54-16.55 (1989); Dower et al., (1988); Taketo (1988)). Thus,previous attempts to improve the electroporation efficiency have focusedon these factors and thus, have primarily involved manipulation ofmethods used to prepare the cells, e.g., washing and centrifugation ofcells during the processing stage, and methods for applying theelectrical shock (i.e., different configuration of the apparatus thatdelivers the electrical pulse).

[0009] Typically, researchers have only modified the host cellsuspension materials to aid in freezing the cells before the electricaltreatment (Taketo 1988).

SUMMARY OF THE INVENTION

[0010] This invention provides improved methods of electroporation andother electrical treatment of cells. The methods comprise the additionof sugars or sugar derivatives, e.g., sugar alcohols, to host cellssuspended in a substantially non-ionic solution, either prior to initialfreezing, or after thawing, but prior to electrotransformation. Themethods of this invention improve electroporation efficiency. The levelof improvement is 30% (for cells that generally exhibit higherefficiency) to 300% (for cells that have lower efficiency).

[0011] In certain embodiments, at least one sugar or sugar derivative isadded to the host cells suspended in a substantially non-ionic solutionprior to electrically treating the host cells. Preferably, the sugar orderivative thereof is added in a concentration range of about 0.1% toabout 5%. In certain embodiments of the invention, the host cells aresuspended in the non-ionic sugar or sugar derivative solution beforethey are electrically transformed. In certain embodiments, one mayprefer to freeze the cells prior to the electrotransformation Forinstance, one may suspend the host cells in the sugar or sugarderivative solution before freezing the cells. In another instance, thecells may be suspended in the sugar or sugar derivative solution afterthe cells have been frozen and thawed. In certain embodiments, the hostcells are bacterial cells, preferably gram-negative bacterial cells, andmost preferably, E. coli.

[0012] This invention also provides a kit for use in the practice of theabove-described methods of transferring nucleic acids of interest intohost cells. The electroporation kit includes host cells suspended in asubstantially non-ionic solution comprising at least one sugar or sugarderivative. In certain embodiments, the kit includes host cellssuspended in a non-ionic solution having a sugar or sugar derivative aconcentration of about 0.1% to about 5%. In other embodiments, the kitincludes a non-ionic solution comprising a mixture of sugars and sugarderivatives.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Thus, this invention provides methods for transferring nucleicacids of interest into host cells, comprising the steps of mixing hostcells suspended in a substantially non-ionic solution comprising atleast one sugar or sugar derivative with nucleic acids of interest, andsubjecting the mixture to electrical treatment, thereby permitting thetransfer of the nucleic acids of interest into the host cells.

[0014] In certain embodiments of the invention, the non-ionic solutionincludes glycerol or dimethyl sulfoxide.

[0015] As used herein, the term “non-ionic solution” refers to a buffersolution that would have minimal or no ions present. In many instances,non-ionic solutions are also non-polar, therefore, for the purposes ofdefining terms in this application, solutions that are non-polar arealso, non-ionic. The concentration of ions in the buffer is adequatelylow so that when electricity is discharged into the host cells, littleor no additional current is carried into the cells. The presence of ionsin the buffer may result in additional current being carried into thecell and can lower the survival rate of the host cells.

[0016] A number of sugars and sugar derivatives are known to thoseskilled in the art. The sugars or sugar derivatives useful in theprocesses and kits of this invention may be in either theD-stereoisomeric or the L-forms (enantiomers) form. Sugars that may beused in the methods and kits of this invention include, but are notlimited to: aldoses, such as monosaccharides which include trioses (i.e.glyceraldehyde), tetroses (i.e. erythrose, threose), pentoses (i.e.arabinose, xylose, ribose, lyxose), hexoses (i.e. glucose, mannose,galactose, idose, gulose, altrose, allose, talose), heptoses (i.e.sedoheptulose), octoses (i.e. glycero-D-manno-octulose), pentose ringsugars (i.e. ribofuranose, ribopyranose); disaccharides (i.e., sucrose,lactose, trehalose, maltose, cellobiose, gentiobiose); andtrisaccharides (i.e., raffinose), oligosaccharides (i.e., amylose,amylopectin, glycogen).

[0017] Sugar derivatives that may be used in the methods and kits ofthis invention include, but are not limited to: alditols or aldosealcohol, which include erythritol, glucitol, sorbitol, or mannitol;ketoses, e.g., dihydroxyacetone, erythrulose, ribulose, xylulose,psicose, fructose, sorbose, and tagatose; aminosugars such asglucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine,muramic acid, N-acetyl muramic acid, and N-acetyineuraminic acid (sialicacid); glycosides, such as glucopyranose and methyl-glucopyranose; andlactones, such as gluconolactone.

[0018] In certain embodiments of the invention, the non-ionic host cellbuffer solution may include a mixture of sugars and derivatives thereof.One skilled in the art would be able to select suitable sugars to placein the mixture and to determine appropriate concentrations of thesesugars and sugar derivatives to optimize the invention.

[0019] In certain embodiments of the methods and kits of this invention,at least one sugar or derivative thereof is added to host cellssuspended in a non-ionic buffer solution prior to electrical treatmentof the cells, wherein the concentration of the sugar or derivativethereof is in the range of about 0.1% to about 5%. Preferably, theconcentration of the sugar or derivative thereof is about 2.0% to about2.5%. In specific embodiments, the added sugar derivative is sorbitoland its concentration is about 2.5%.

[0020] As used herein, the term “electrical treatment” includes anymethod of using electrical pulses or electrical discharges to introducegenes, fragments of genes, or other nucleic acids of interest into acell. Electroporation methods are well known to those skilled in the art(See, e.g., Sambrook et al. 1987; Stratagene Instruction Manual forEpicurian Coli® Electroporation-Competent Cells 1997). Conditions foroptimal efficiency can be determined by one skilled in the art.

[0021] In certain embodiments of the invention, bacterial cells aresuspended in the non-ionic buffer solution comprising at least one sugaror sugar derivative prior to electrical treatment. Preferably, thebacterial cells are gram-negative bacterial cells (Davis, B. D et al.,Microbiology: 3^(rd) Edition (eds. Davis, B. D. et al. Harper and Row,1995)). In a preferred embodiment of the invention, the gram-negativebacterial cells are E. coli. In a specific embodiment, the bacterialcell strain is XL1-Blue™ (Stratagene Catalogue #200268).

[0022] As used herein, the term “nucleic acids of interest” includes,but is not limited to, nucleic acid sequences that encode functional ornon-functional proteins, and fragments of those sequences,polynucleotides, or oligonucleotides. The nucleic acids of interest maybe obtained naturally or synthetically, e.g., using PCR or mutagenesis.Further, the nucleic acids may be circular, linear, or supercoiled intheir topology. Preferably, the nucleic acids are linear. Although notlimited to such sizes, certain embodiments of this invention employnucleic acids of interest ranging from about 3 kb to about 300 kb,depending on factors well known to those of skill in the art.

[0023] As used herein, the term “permitting the transfer of the nucleicacids of interest into the cells” may include transient transfer orpermanent incorporation of the nucleic acids of interest into thebacterial cells by either autonomous replication or integration into thegenome. One skilled in the art would be able to determine the optimalconditions to transfer the nucleic acids of interest, e.g., length ofnucleic acids, pulse time. Typically, one skilled in the art may subjectthe host cells to electroporation for a certain period of time, therebyinsuring the transfer of nucleic acids, but possibly sacrificing a largenumber of cells. This invention allows a larger number of host cellssuspended in the above-described solutions to survive electroporationthan cells suspended in the previously known solutions. Typically,subjecting E. coli to electrical transformation caused >90% of the cellsto die; however, by practicing the methods of this invention, more ofthe cells will survive.

[0024] One skilled in the art would know how to select an appropriatemedia to promote growth of the transformed cells. The chosen mediashould propagate the transformed cells that either transiently expressor have nucleic acids integrated into the host cells' genome. Further,the media should be selected so as to assist the cells in recoveringfrom the electrical treatment.

[0025] This invention also provides kits used in the practice of themethods of transferring nucleic acids into bacterial host cellsaccording to this invention. In certain embodiments, the kits comprisetransformation competent host cells suspended in a substantiallynon-ionic solution comprising at least one sugar or sugar derivative. Incertain embodiments the kits, the concentration of the sugar orderivative thereof is in the range of about 0.1% to about 5%. In certainembodiments of the kits of this invention, the transformation competenthost cells are bacterial cells, preferably E. coli. Other contents ofthe kit may include a control plasmid DNA for use in determining whethertransformation has occurred.

[0026] The invention described above may be better understood byreference to the following examples. However, these examples are offeredsolely for the purpose of illustrating the invention, and should not beinterpreted as limiting the invention.

EXAMPLES

[0027] Prior to electroporation, E. coli cells are usually grown inenriched media and then, suspended in 10% glycerol. The glycerol servesat least two purposes. It is non-polar and thus, does not carry excesselectrical charge into the host cells. In addition, the transformationcompetent host cells typically are stored at temperatures below −70° C.Cell viability during this freezing process is improved if cells aresuspended in cryopreservative compounds, e.g., glycerol, dimethylsulfoxide.

[0028] Fermentation and Inoculation

[0029] A 15 liter Applikon™ fermenter was thoroughly cleaned withreverse osmosis water. After cleaning, liquid remaining in the fermenterwas removed to provide an ion-free environment. Specifically, all tracesof magnesium were removed from the fermenter. Media to grow the E. coliwas produced in the glass vessel of the fermenter by adding 240-280grams DIFCO™ tryptone, 60-70 grams of DIFCO™ yeast, and 6 grams of NaClto the empty vessel. 12 L of 0.2 micro filtered water was added to thefermenter. The fermenter was autoclaved on slow exhaust for 35-45minutes and cooled. After removing the fermenter from the autoclave, itwas heated up to 37° C.

[0030] XL1-Blue (Stratagene Catalogue #200228) was streaked out on Tetplates and incubated at 37° C. for 24 hours (Sambrook et al. 1987).Since this method may be used to prepare different types ofelectrocompetent cells, one skilled in the art would know on whichplates the different types of cells grow. For example, SURE™(Stratagene) and XL1-Blue MRF′™ (Stratagene) should be streaked out onTet plates, TG-1™ on LB plates.

[0031] Under sterile conditions, 75-100 ml of magnesium-free SOB (20grams Tryptone, 5 grams yeast extract, and 0.5 grams of NaCl per liter)was poured into three sterile 250 ml flasks. 2-5 bacterial colonies,between 2-5 mm in width, were chosen and inoculated into themagnesium-free SOB. The cells were grown overnight at room temperaturein an air shaker at 250-275 rpm.

[0032] The optical density (O.D.) of the cells cultured overnight wasdetermined with a Beckman DU640B spectrometer by diluting the cells 1 to10, i.e., 900 μl of media was added to a quartz cuvette, thespectrometer was zeroed with the media-filled cuvette, 100 μl of cellswas added to the cuvette, and the O.D. of the cells was determined.

[0033] Under sterile conditions, 60-100 O.D. units of the cells wereadded into the fermenter using an electric pump. The temperature of thefermenter was maintained at 37° C. The temperature to set the fermentermay vary depending on what cells are used. For XL1-Blue™, XL1-BlueMRF′™, and TG-1™ cells, the fermenter should be set at 37° C., and forSURE™ cells, the machine should be set at 38.5-39° C. Those skilled inthe art know how to determine the optimal conditions for growingdifferent bacterial cells in a fermenter.

[0034] When the O.D. of the fermenter is 0.15-0.25 or when it was nolonger possible to see through the fermenter, as much SOB as thefermenter would hold and 1-2 drops of sterile anti-foam A™ (Sigma) wereadded. To maintain oxygen content, the culture was agitated with airing.The fermentation process took about five hours. One skilled in the artcould easily determine the optimal conditions and time frame forharvesting the cells.

[0035] At the desired final O.D. 0.82 [Beckman DU640B spec 550 nm], thefermenter was cooled to 4° C. The bacteria was concentrated to 0.5liters with a mini-sert crossflow filtration unit from Sartorious. Whenthe fluid level inside the fermenter was 0.75 liters, a buffer exchangewas set up. The buffer exchange was run until 2.0 gallons of sterilecooled water (4° C.) had been exchanged. Another buffer exchange was runwith 1.0 gallon of pre-cooled 0.2 micro filtered water+15% glycerol.

[0036] Afterwards, the cells were removed from the fermenter. The cellswere split into 2 spin buckets and centrifuged (Sorval Centrifuge™) for15 minutes at 4,000 rpm at 0° C. The supernate was decanted. 35 ml of15% glycerol/water was poured into the spin buckets and the pellets werepipetted into solution as quickly as possible. After solubilizing thepellet, the solutions were combined into one spin bucket. The cells werealiquoted in 100 μl batches and placed in a −80° C. freezer for storage.

[0037] Additives Added to the Competent Bacterial Cell Preparation

[0038] The effect of non-polar aldoses and aldose alcohols was tested tosee if those compounds improved the electroporation efficiency in E.coli. Specifically, galactose (an aldohexose monosaccharide), maltose(an aldohexose disaccharide), mannitol (an aldohexose alcohol), andsorbitol (an aldohexose alcohol) additives were tested.

[0039] After two hours of freezing, competent cells were thawed andamounts varying between 0.1% and 5.0% of various sugar or sugar aldoseswere added to the thawed cells. 1 μl of 10 pg/μl plasmid, pCMV-Script(Stratagene #211199) in TE buffer or water (or other low-ionic strengthbuffer) was added to 40 pi of the treated cells in a chilled 1.5 mlMicrofuge tube. The cells were gently mixed and chilled.

[0040] The DNA-cell mixture was transferred to a chilled electroporationcuvette. The cuvette was pulsed once, using a Bio-Rad II Gene Pulsar, at1.7 kV (kilo Volts), 200 Ω and 25 μF. The cuvette was immediatelyremoved and 960 μl of SOC medium (2 ml of 20% glucose and 1 ml of 2M Mgper 100 ml of SOB medium) was added to resuspend the cells.

[0041] That suspension was transferred to a sterile 15-ml Falcon 2059polypropylene tube and shaken at 225-250 rpm at 37° C. for 1 hour. Wepipetted 2.5 μl of transformed cells into a 100 μl pool of SOC medium onan Amp plate (Sambrook et al. 1997). The cell/media mixture was spreadover the Amp plates. The plates were incubated overnight (16-20 hours)at 37° C.

[0042] The treated cells demonstrated an improvement in transferefficiency. See Table 1.

[0043] Sorbitol Concentrations Added to Competent Bacterial CellPreparation

[0044] The effect of adding sugars, prior to freezing the cells, wastested for improved electroporation efficiency. Sorbitol was chosen asthe first test compound.

[0045] To determine the optimal concentration of sorbitol, the aboveprotocol was followed to produce electrocompetent bacterial cells.However, before freezing the cells, a solution of sorbitol was addedsuch that the final concentrations of the suspension of cells variedfrom 0%, 1.0%, 2.0%, and 2.5%.

[0046] 1 μl of 10 pg/μl plasmid, pUC18 (New England Biolabs) in TEbuffer or water (or other low-ionic strength buffer), was added to 40 μlof the treated cells in a chilled 1.5 ml Microfuge tube. Transformedcells were obtained by following the above protocol. Various experimentswere run to determine the efficiency of transformation. Five differentpreparations were tested in triplicate, and each preparation was platedin duplicate. See Table 2. TABLE 1 The Effect of Different Additives onBacterial Cells in Electroporation Experiments Additive finalconcentration Relative efficiency* None — 1.00 galactose  0.5% 2.20 1.0% 1.50  2.0% 2.40 maltose  1.0% 1.50  2.5% 3.00  3.0% 1.30  4.0%1.50 mannitol 0.75% 3.00 1.00% 2.80 1.25% 3.30 2.00% 2.90 2.20% 2.802.40% 1.90 sorbitol 0.75% 2.20 1.00% 2.80 1.50% 3.10 2.00% 1.50

[0047] TABLE 2 EFFECT OF SORBITOL ADDITIVE ON XL1 - BLUE CELLS INELECTROTRANSFORMATION Percent sorbitol Efficiency (cfu/ug pUC18)Experiment #1   0% 9.0 × 10⁹   1.0% 9.3 × 10⁹   2.0% 1.2 × 10¹⁰ 2.5% 1.6× 10¹⁰ Experiment #2   0% 8.72 × 10⁹   1.0% 1.23 × 10¹⁰ 2.0% 1.66 × 10¹⁰2.5% 1.48 × 10¹⁰ Experiment #3   0% 9.26 × 10⁹   1.0% 1.03 × 10¹⁰ 2.0%1.23 × 10¹⁰ 2.5% 1.33 × 10¹⁰ Experiment #4   0% 7.10 × 10⁹   1.0% 9.00 ×10⁹   2.0% 1.29 × 10¹⁰ 2.5% 1.39 × 10¹⁰ Experiment #5   0% 8.89 × 10⁹  1.0% 9.27 × 10⁹   2.0% 1.60 × 10¹⁰ 2.5% 1.60 × 10¹⁰

[0048] All documents referenced in this application, including but notlimited to, articles, books, reviews, patents, and patent applications,are hereby incorporated by reference in their entirety into thisspecification.

ADDITIONAL REFERENCES

[0049] 1. Greener, A., Strategies, 6:7-9 (1993).

[0050] 2. Greener, A., Strategies, 3:5-6 (1990).

[0051] 3. Bullock, W. O., Fernandex, J. M., and Short, J. M.,Biotechniques, 5:81-83 (1987).

[0052] 4. Jerpseth, B., Greener, A., Short, J. M., Viola, J., and Kretz,P. L., Strategies, 5:81-83 (1992).

What is claimed is:
 1. A method for transferring nucleic acids ofinterest into competent host cells, comprising the steps of: (a) mixingcompetent host cells suspended in a substantially non-ionic solutioncomprising at least one sugar or sugar derivative with the nucleic acidsof interest; and (b) subjecting the host cells to an electricaltreatment, thereby permitting the transfer of the nucleic acids ofinterest into the bacterial cells.
 2. The method of claim 1, wherein thenon-ionic solution further comprises glycerol or dimethyl sulfoxide. 3.The method of claim 1, wherein the host cells are gram-negativebacterial cells.
 4. The method of claim 3, wherein the gram-negativebacterial cells are E. coli.
 5. The method of 1, further comprising thestep of culturing the transformed cells in a selected media capable ofpromoting their growth.
 6. The method according to claim 1, wherein theconcentration of the sugar or derivative is in the range of about 0.1%to about 5%.
 7. The method according to claim 1, wherein the sugar orsugar derivative is sorbitol in a concentration range of about 2.0 toabout 2.5%.
 8. The method according to claim 1, wherein the sugar orsugar derivative is an aldose.
 9. The method according to claim 8,wherein the aldose is selected from the group consisting ofmonosaccharides, disaccharides, trisaccharides, and oligosaccharides.10. The method according to claim 1, wherein the sugar or sugarderivative is an aldose alcohol.
 11. The method according to claim 10,wherein the aldose alcohol is selected from the group consisting oferythritol, sorbitol, and mannitol.
 12. The method according to claim 1,wherein the sugar or sugar derivative is a ketose.
 13. The methodaccording to claim 12, wherein the ketose is selected from the groupconsisting of dihydroxyacetone, erythrulose, ribulose, xylulose,psicose, fructose, sorbose, and tagatose.
 14. The method according toclaim 1, wherein the sugar or sugar derivative is an aminosugar.
 15. Themethod according to claim 14, wherein the aminosugar is selected fromthe group consisting at least one of glucosamine, galactosamine,N-acetylglucosamine, N-acetylgalactosamine, muramic acid, N-acetylmuramic acid, and sialic acid.
 16. The method according to claim 1,wherein the sugar or sugar derivative is a glycoside.
 17. The methodaccording to claim 16, wherein the glycoside is selected from the groupconsisting of glucopyranose and methyl-glucopyranose.
 18. The methodaccording to claim 1, wherein the sugar or derivative thereof is alactone.
 19. The method according to claim 18, wherein the lactone isgluconolactone.
 20. The method according to claim 1, wherein thenon-ionic solution comprises a mixture of sugars and sugar derivatives.21. An electroporation kit comprising transformation competent cellssuspended in a substantially non-ionic solution comprising at least onesugar or sugar derivative.
 22. The kit according to claim 21, whereinthe transformation competent cells are gram-negative bacterial cells.23. The kit according to claim 21, wherein the bacterial cells are E.coli.
 24. The kit according to claim 21, wherein the concentration ofthe sugar or derivative thereof is in the range of about 0.1% to about5%.
 25. The kit according to claim 1, wherein the sugar or sugarderivative is sorbitol in a concentration range of about 2.0 to about2.5%.
 26. The kit according to claim 21, wherein the sugar or sugarderivative is an aldose.
 27. The kit according to claim 26, wherein thealdose is selected from the group consisting of monosaccharides,disaccharides, trisaccharides, and oligosaccharides.
 28. The kitaccording to claim 21, wherein the sugar or sugar derivative is analdose alcohol.
 29. The kit according to claim 28, wherein the aldosealcohol is selected from the group consisting of erythritol, sorbitol,and mannitol.
 30. The kit according to claim 21, wherein the sugar orsugar derivative is a ketose.
 31. The kit according to claim 30, whereinthe ketose is selected from the group consisting of dihydroxyacetone,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, andtagatose.
 32. The kit according to claim 21, wherein the sugar or sugarderivative is an aminosugar.
 33. The kit according to claim 32, whereinthe aminosugar is selected from the group consisting at least one ofglucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine,muramic acid, N-acetyl muramic acid, and sialic acid.
 34. The kitaccording to claim 21, wherein the sugar or sugar derivative is aglycoside.
 35. The kit according to claim 34, wherein the glycoside isselected from the group consisting of glucopyranose andmethyl-glucopyranose.
 36. The kit according to claim 21, wherein thesugar or derivative thereof is a lactone.
 37. The kit according to claim36, wherein the lactone is gluconolactone.
 38. The kit according toclaim 21, wherein the non-ionic solution comprises a mixture of sugarsand sugar derivatives.